Eukaryotic mechanosensory transduction channel

ABSTRACT

The present invention provides, for the first time, nucleic acids encoding a eukaryotic mechanosensory transduction channel (MSC) protein. The proteins encoded by these nucleic acids form channels that can directly detect mechanical stimuli and convert them into electrical signals. These nucleic acids and the proteins they encode can be used as probes for sensory cells in animals, and can be used to diagnose and treat any of a number of human conditions involving inherited, casual, or environmentally-induced loss of mechanosensory transduction activity.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. DC03160,awarded by the National Institutes of Health. The Government has certainrights in this invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

Not applicable.

FIELD OF THE INVENTION

This invention provides isolated nucleic acid and amino acid sequencesof a novel family of eukaryotic mechanosensory ion channels that aredesignated mechanosensory transduction channels (MSC).

BACKGROUND OF THE INVENTION

The ability to detect mechanical stimuli is an essential and prevalentcharacteristic of living organisms, and is found from bacteria to simplemetazoans to the most complex of mammals. Indeed, the ability to detectmechanical stimuli and convert them into electrical signals forms thebasis of many central aspects of animal life, such as light touch, heavytouch, proprioception, baroreception, balance, and the crown jewel,hearing. Even the ability of cells to stop growing when in contact withneighboring cells is likely dependent on mechanical stimuli. Notsurprisingly, therefore, numerous human conditions result at least inpart from an inability to detect mechanical stimuli, such as Meniere'sDisease, sensorineural deafness, blood pressure disorders, and varioustypes of cancers.

In general, the variety of known mechanosensory modalities are thoughtto be mediated by mechanically-gated cation channels present within themembrane of receptor cells. This view has come in large part fromdetailed studies into the physiology of mechanosensation using variouscell types involved in mechanosensory detection, such as the hair cellsof the vertebrate inner ear, single-celled ciliates such as Paramecium,or the sensory neurons of Drosophila (see, e.g., Kernan et al., Neuron12:1195-1206 (1994)). In Drosophila, the dendrite of the sensory neuronis enclosed in a cavity filled with a specialized receptor lymph, whichis unusually rich in potassium ions, and is functionally equivalent tothe potassium-rich endolymph of the vertebrate cochlea. These potassiumions produce a transepithelial potential difference, with the apicalside of the epithelium being positively charged. Mechanical stimulationof the bristle, which is adjacent to the sensory neuron, generates amechanoreceptor potential within the neuron, detectable as a negativedeflection of the transepithelial potential, which reflects the flow ofcations from the receptor lymph into the sensory neuron.

Activation of the hair cells of vertebrates also result in the influx ofcations into cells (see, e.g., Hudspeth, Nature, 341:397-404 (1989)).Each hair cell has a number of specialized microvillar structures,called stereocilia, whose deflection results in the activation of aputative channel present on the surface of the cell. Interestingly,electrophysiological studies have suggested that these cells contain asimilar number of receptor channels as they do stereocilia, suggestingthat perhaps each receptor channel is coupled to a single stereocilium.In addition, studies of the kinetics of hair-cell activation havesuggested that the putative mechanosensory receptors are directlystimulated by mechanical force, resulting in the direct opening of thechannel without the involvement of second messengers.

Despite the great importance of mechanosensation for animal behavior andhealth, and the detailed electrophysiological understanding that hasbeen gained from the above-described studies, almost nothing is knownabout the molecular basis of mechanosensory detection in eukaryotes.Several mutations and distantly related molecules involved in thisprocess have, however, been found. In Drosophila, for example, a numberof mutations have been isolated that disrupt mechanoreception, resultingin a variety of phenotypes such as reduced locomotor activity, totaluncoordination, and even death (Kernan et al., Neuron 12:1195-1206(1994)). Also, mutations have been identified in the nematode C. elegansthat result in a loss of sensitivity to gentle touch (reviewed inGarcia-Aanoveros & Corey, Ann. Rev. Neurosci. 20:567-594 (1997)). Inaddition, a prokaryotic mechanosensory channel has been identified(Sukarev et al., Nature 368:265-268 (1994)). Still, despite theseadvances, the principle molecule of the mechanosensory transductionprocess in eukaryotes, the mechanically gated channel, has yet to beisolated or identified.

The identification and isolation of eukaryotic mechanosensorytransduction channels would allow for the development of new methods ofpharmacological and genetic modulation of mechanosensory transductionpathways. For example, availability of mechanosensory transductionchannel proteins would permit screening for high-affinity agonists,antagonists, and modulators of mechanosensation in animals. Suchmolecules could then be used, e.g., in the pharmaceutical industry, totreat one or more of the many human conditions involving loss orhyperactivation of mechanosensation. In addition, the determination ofnucleotide and amino acid sequences of mechanosensory transductionchannels associated with a human condition would provide new tools forthe diagnosis and/or treatment, e.g., gene-based treatment, of thecondition.

SUMMARY OF THE INVENTION

The present invention provides for the first time nucleic acids encodinga eukaryotic mechanosensory transduction protein. The nucleic acids andthe polypeptides they encode are referred herein as mechanosensorychannel (MSC) nucleic acids and proteins. In vivo, MSC proteins formmechanosensory transduction channels that play a central role in manycritical processes such as hearing, proprioception, and tactilesensation.

In one aspect, the present invention provides an isolated nucleic acidencoding a mechanosensory transduction protein, the protein having atleast one of the following characteristics: (i) comprising greater thanabout 70% amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4;(ii) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii)specifically binding to polyclonal antibodies generated against apolypeptide comprising an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:4; wherein the protein does not comprise the polypeptide sequence ofSEQ ID NO:6.

In one embodiment, the nucleic acid encodes a polypeptide comprising theamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In anotherembodiment, the nucleic acid comprises a nucleotide sequence of SEQ IDNO:1 or SEQ ID NO:3, but not SEQ ID NO:5.

In another embodiment, the nucleic acid selectively hybridizes undermoderately stringent wash conditions to a nucleic acid comprising anucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. In anotherembodiment, the nucleic acid selectively hybridizes under stringent washconditions to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:1 or SEQ ID NO:3, but not SEQ ID NO:5.

In another embodiment, the nucleic acid is amplified by primers thatselectively hybridize under stringent hybridization conditions to thesame sequence as degenerate primer sets encoding an amino acid sequenceselected from the group consisting of: LDVLIENEQKEV (SEQ ID NO:7),HHLFGPWAIII (SEQ ID NO:8), and VLINLLIAMMSDTYQRIQ (SEQ ID NO:9).

In another embodiment, the nucleic acid is less than 120 kb. In anotherembodiment, the nucleic acid is less than 90 kb. In another embodiment,the nucleic acid is less than 60 kb. In another embodiment, the nucleicacid is less than 30 kb. In another embodiment, the nucleic acid is lessthan 10 kb. In another embodiment, the nucleic acid sequence encodingthe MSC protein is isolated away from its genomic neighbors.

In another aspect, the present invention provides an expression cassettecomprising a nucleic acid encoding a mechanosensory transductionprotein, the protein having at least one of the followingcharacteristics: (i) comprising greater than about 70% amino acidsequence identity to SEQ ID NO:2 or SEQ ID NO:4; (ii) comprising anamino acid sequence selected from the group consisting of SEQ ID NO:7,SEQ ID NO:8, and SEQ ID NO:9; or (iii) specifically binding topolyclonal antibodies generated against a polypeptide comprising anamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; wherein the proteindoes not comprise the polypeptide sequence of SEQ ID NO:6.

In another aspect, the present invention provides an isolated eukaryoticcell comprising the expression cassette.

In one aspect, the present invention provides an isolated nucleic acidencoding an extracellular domain of a mechanosensory transductionprotein, the extracellular domain comprising greater than about 70%amino acid sequence identity to an extracellular domain of SEQ ID NO:2or SEQ ID NO:4, wherein the extracellular domain does not comprise anextracellular domain of SEQ ID NO:6.

In one embodiment, the extracellular domain is fused to a heterologouspolypeptide, thereby forming a chimeric polypeptide. In anotherembodiment, the extracellular domain comprises an amino acid sequence ofan extracellular domain of SEQ ID NO:2 or SEQ ID NO:4.

In another aspect, the present invention provides an isolatedmechanosensory transduction protein, the protein having at least one ofthe following characteristics: (i) comprising greater than about 70%amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4; (ii)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii) specifically bindingto polyclonal antibodies generated against a polypeptide comprising anamino acid sequence of SEQ ID NO:2, or SEQ ID NO:4; wherein the proteindoes not comprise the amino acid sequence of SEQ ID NO:6.

In one embodiment, the protein comprises the amino acid sequence of SEQID NO:2 or SEQ ID NO:4.

In another aspect, the present invention provides an isolatedpolypeptide comprising an extracellular domain of a mechanosensorytransduction protein, the extracellular domain comprising greater thanabout 70% amino acid sequence identity to an extracellular domain of SEQID NO:2 or SEQ ID NO:4, wherein the extracellular domain does notcomprise the amino acid sequence of an extracellular domain of SEQ IDNO:6.

In one embodiment, the extracellular domain is fused to a heterologouspolypeptide, forming a chimeric polypeptide. In another embodiment, theextracellular domain comprises the amino acid sequence of anextracellular domain of SEQ ID NO:2 or SEQ ID NO:4.

In another aspect, the present invention provides an antibody thatselectively binds to a mechanosensory transduction protein, the proteinhaving at least one of the following characteristics: (i) comprisinggreater than about 70% amino acid sequence identity to SEQ ID NO:2 orSEQ ID NO:4; (ii) comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii)specifically binding to polyclonal antibodies generated against apolypeptide comprising an amino acid sequence of SEQ ID NO:2, or SEQ IDNO:4; wherein the protein does not comprise the amino acid sequence ofSEQ ID NO:6.

In another aspect, the present invention provides a method foridentifying a compound that modulates mechanosensory receptor activityin eukaryotic cells, the method comprising the steps of: (i) contactingthe compound with a mechanosensory receptor protein, the protein havingat least one of the following characteristics: (a) comprising greaterthan about 70% amino acid sequence identity to a sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; (b)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (c) specifically bindingto polyclonal antibodies generated against a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4, and SEQ ID NO:6; and (ii) determining the functional effectof the compound on the mechanosensory receptor protein.

In one embodiment, the mechanosensory receptor protein is expressed in aeukaryotic cell or cell membrane. In another embodiment, the functionaleffect is determined by detecting a change in the mechanoreceptorpotential of the cell or cell membrane. In another embodiment, thefunctional effect is determined by detecting a change in anintracellular ion concentration. In another embodiment, the ion isselected from the group consisting of K⁺ and Ca²⁺. In anotherembodiment, the protein comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. Inanother embodiment, the protein is recombinant. In another embodiment,the functional effect is a physical interaction with the receptorprotein.

In another aspect, the present invention provides a method of genotypinga human for a mechanosensory transduction channel locus, the methodcomprising detecting a mutation in a nucleic acid encoding amechanosensory transduction channel in the human, the protein having atleast one of the following characteristics: (a) comprising greater thanabout 70% amino acid sequence identity to a polypeptide having asequence of SEQ ID NO:2; (b) having greater than about 90% amino acidsequence identity to a polypeptide having a sequence of SEQ ID NO:5; (c)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; or (d) specifically bindingto polyclonal antibodies generated against a polypeptide selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, and SEQ ID NO:8; wherein the mutation introduces a premature stopcodon into the nucleic acid 5′ to the transmembrane domain region of theprotein, or is a missense mutation removing a cysteine residue betweentransmembrane segments 4 and 5 of the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment between Drosophila melanogaster andCaenorhabditis elegans MSC homologs.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The present invention provides, for the first time, nucleic acidsencoding a eukaryotic mechanosensory transduction channel (MSC) protein.Mutations in these nucleic acids and the proteins they encode areresponsible for the “no-mechanoreceptor potential” phenotype inDrosophila, a phenotype involving uncoordination, often to the point oflethality, and a loss of mechanoreceptor potential in the bristles ofmutant flies (Kernan et al., Neuron 12:1195-1206 (1994)). The proteinsencoded by these nucleic acids form channels (e.g., as tetramers) thatcan directly detect mechanical stimuli and convert them into electricalsignals. These proteins can detect mechanical stimuli in any of a numberof sensory cells, such as neuronal sensory cells, hair cells, andothers. These nucleic acids and the proteins they encode can be used asprobes for sensory cells in animals, and can be used to diagnose andtreat any of a number of human conditions involving inherited, casual,or environmentally-induced loss of mechanosensory transduction activity.

The present invention also provides methods of screening for modulators,e.g., activators, inhibitors, enhancers, etc., of mechanosensorytransduction channels. Such modulators would be useful to altermechanosensory transduction activity in an animal, e.g., for thetreatment of any of a number of human disorders. Thus, the inventionprovides assays for mechanosensory transduction modulation, where theMSC proteins act as a direct or indirect reporter for mechanosensorytransduction activity. MSC proteins can be used in assays, in vitro, invivo, or ex vivo, to detect changes in ion flux, ion concentration,membrane potential, signal transduction, transcription, or otherbiological or biophysical effects of mechanical stimulus detection.

In one embodiment, MSC proteins can be used as indirect reporters viaattachment to a second reporter molecule such as green fluorescentprotein (see, e.g., Mistili & Spector, Nature Biotechnology, 15:961-964(1997)). In one embodiment, MSC proteins are recombinantly expressed incells, e.g., Xenopus oocytes, and modulation of mechanosensorytransduction is assayed by detecting changes in transmembrane potential,mechanosensory potential, intracellular ion concentration, ion flux, andthe like.

In certain embodiments, potential modulators are identified by virtue ofan ability to physically interact with an MSC protein. Assays forphysically-interacting molecules would provide an efficient primaryscreen for candidate MSC modulators, and, in addition, would allow theidentification of proteins and other compounds that naturally interactwith MSC proteins in vivo.

The invention also provides methods of detecting MSC nucleic acid andprotein expression, allowing investigation into mechanosensoryregulation and the identification of mechanosensory cells. The presentnucleic acids and proteins can also be used to genotype an animal,including humans, for forensic, paternity, epidemiological, or otherinvestigations. The present invention also provides conserved sequencesfound in multiple MSC sequences, allowing the identification of evendistantly related MSC homologs (see, for example, SEQ ID NOs:7-9). Inaddition, the present invention provides methods for identifyingmutations in a mechanosensory transduction channel protein thateliminate or reduce function of the channel. Such mutations likelyunderlie one or more of the human conditions involving loss ofmechanosensation discussed herein. As such, the invention providesmethods of diagnosing mechanosensory transduction defects in animals.

Functionally, the MSC proteins form, within a cell membrane, a channelthat directly detects mechanical stimuli and, in response to thestimuli, allows the influx of cations into a cell, thereby depolarizingthe cell and initiating an electrical, i.e. neural, signal.

Structurally, the nucleotide sequences of MSCs (see, e.g., SEQ ID NOs:1, 3, and 5, representing the Drosophila genomic, Drosophila cDNA, andCaenorhabditis elegans genomic sequences, respectively) encodepolypeptides of from about 1619-1709 amino acids with a predictedmolecular weight of about 177 kDa (see, e.g., SEQ ID NOs:2, 4, and 6).The MSC genes typically contain about 19 exons, encoding a protein withabout 27 ankyrin repeats and from 6-11, typically about 8, transmembranedomains. Such proteins are weakly related to the TRP family ofepithelial cation channels. MSC homologs from other species typicallyshare at least about 70% identity over a region of at least about 25amino acids in length, preferably 50 to 100 amino acids in length.

The present invention provides nucleic acids comprising an MSC whereinthe nucleic acid is less than 120, 90, 60, 30, 20, 10, or 7 kb. Inaddition, nucleic acids comprising MSCs are provided wherein the MSCpolynucleotide is isolated away from its genomic neighbors, i.e., thenucleic acid does not comprise any genes that are located within thesame genomic region as the MSC gene.

The present invention also provides polymorphic variants of the MSCdepicted in SEQ ID NO:2: variant #1, in which an isoleucine residue issubstituted for a leucine residue at amino acid position 6; variant #2,in which a glycine residue is substituted for an alanine residue atamino acid position 13; and variant #3, in which an arginine residue issubstituted for a lysine residue at amino acid position 22.

The present invention also provides polymorphic variants of the MSCdepicted in SEQ ID NO:4: variant #1, in which an isoleucine residue issubstituted for a leucine residue at amino acid position 24; variant #2,in which an alanine residue is substituted for a glycine residue atamino acid position 26; and variant #3, in which an aspartic acidresidue is substituted for a glutamic acid residue at amino acidposition 30.

The present invention also provides mutated MSC sequences that eliminatemechanosensory transduction activity in vivo. For example, mutationsthat prematurely truncate MSC proteins in the ankyrin repeat region, ormissense mutations that alter a cysteine residue between transmembranesegments four and five, e.g., a C to Y substitution, have beendiscovered that eliminate or severely reduce MSC activity. Suchmutations can be used, e.g., to detect defects in mechanosensation,specifically in mechanosensory transduction channels, in an animal suchas a human.

Specific regions of MSC may be used to identify polymorphic variants,interspecies homologs, and alleles of MSC. Such identification can bemade in vitro, e.g., under stringent hybridization conditions or by PCR(e.g., using primers encoding SEQ ID NOs 7-9) and sequencing, or byusing the sequence information provided herein in a computer system forcomparison with other nucleotide sequences. Typically, identification ofpolymorphic variants and alleles of MSC proteins is made by comparing anamino acid sequence of about 25 amino acids or more, e.g., 50-100 aminoacids. Amino acid identify of approximately at least about 70% or above,preferably 80%, most preferably 90-95% or above typically demonstratesthat a protein is a polymorphic variant, interspecies homolog, or alleleof MSC protein. Sequence comparison can be performed using any of thesequence comparison algorithms discussed herein. Antibodies thatspecifically bind to MSC protein or a conserved region thereof can alsobe used to identify alleles, interspecies homologs, and polymorphicvariants.

Polymorphic variants, interspecies homologs, and alleles of MSC proteinscan be confirmed by examining mechanosensory cell-specific expression ofthe putative MSC homolog. Typically, an MSC protein having a sequence ofSEQ ID NO:2, 4, or 6 can be used as a positive control in comparison tothe putative homolog. Such putative homologs are expected to retain theMSC structure described herein, i.e. intracellular domain with multiple,e.g., 27, ankyrin repeats, and a transmembrane domain containingmultiple, e.g, 8, transmembrane domains.

The present invention also provides promoters, enhancers, 5′- and3′-untranslated regions, and numerous other regulatory elements thatcontrol the transcription, translation, mRNA stability, mRNAlocalization, and other factors regulating MSC expression. For example,SEQ ID NO:1 provides genomic DNA sequence including MSC coding sequenceas well as upstream and downstream regulatory sequences, includingpromoter sequences, etc. Promoters and other regulatory sequences can beidentified using standard methods well known to those of skill in theart, including by homology to well conserved regulatory elements such asthe TATA box or other elements, as taught, e.g., in Ausubel et al.,supra, or in Lewin, Genes IV (1990). Promoter, enhancer, and otherregulatory elements can also be determined functionally, e.g., by fusingspecific regions of SEQ ID NO:1 to a reporter gene and determining whichregions are sufficient for expression of the reporter gene, or bymutagenizing specific regions of SEQ ID NO:1 and thereby determiningwhich regions are required for expression. Such methods are well knownto those of skill in the art. Any of the present regulatory elements canbe used in isolation or together, and can be used to drive theexpression of an MSC protein, a marker protein, or any protein or RNAthat is desirably expressed in a cell or other expression system. Inpreferred embodiments, an MSC regulatory element is used to drive theexpression of a protein, e.g., an MSC or a heterologous polypeptide, ina tissue-specific manner, i.e., specifically in mechanosensory cells.

MSC nucleotide and amino acid sequences can also be used to constructmodels of mechanosensory transduction cell proteins in a computersystem. Such models can be used, e.g., to identify compounds that mayinteract with, activate, or inhibit MSC protein channels. Such compoundscan then be used for various applications, such as to modulatemechanosensory transduction activity in vivo or to investigate thevarious roles of MSC in mechanosensory transduction in vivo.

The isolation of MSC protein also provides a means for assaying forinhibitors and activators of mechanosensory transduction channels, aswell as for molecules, e.g., proteins, that interact with MSC proteinsin vitro or in vivo. Biologically active MSC protein channels are usefulfor testing inhibitors and activators of MSC as mechanosensorytransduction channels using in vivo and in vitro expression, e.g., inoocytes, and measuring MSC expression, phosphorylation state, membranepotential, mechanosensory potential, intra- or extra-cellular ionconcentration, ion flux, and the like. Molecules can also be screenedfor the ability to physically interact with, e.g., bind to, MSCproteins, fragments thereof, or MSC nucleic acids, e.g., MSC promotersequences, as shown in SEQ ID NO:1 and SEQ ID NO:3. Such interactingmolecules can interact with any part of an MSC, e.g., the extracellulardomain, transmembrane domain region, or intracellular domain, e.g., anankyrin repeat. Such molecules may be involved in, or used to identifymolecules capable of modulating, any aspect of MSC activity, includingchannel formation, detection of a mechanical stimulus, opening and/orclosing of the channel, ion specificity of the channel, adaptation ofthe channel, or any other functional or physical aspect of the channel.

The present invention also provides assays, preferably high throughputassays, to identify molecules that interact with and/or modulate an MSCpolypeptide. In numerous assays, a particular domain of an MSC is used,e.g., an extracellular, transmembrane, or intracellular domain. Innumerous embodiments, an extracellular domain is bound to a solidsubstrate, and used, e.g., to isolate enhancers, inhibitors, or anymolecule that can bind to and/or modulate the activity of anextracellular domain of an MSC polypeptide. In certain embodiments, adomain of an MSC polypeptide, e.g., an extracellular, transmembrane, orintracellular domain, is fused to a heterologous polypeptide, therebyforming a chimeric polypeptide. Such chimeric polypeptides are useful,e.g., in assays to identify modulators of an MSC polypeptide.

Such modulators and interacting molecules can be used for variouspurposes, such as to further investigate mechanosensory transductionchannel activity in animal cells, or to modulate mechanosensorytransduction activity in cells, e.g. to treat one or more conditionsassociated with a mechanosensory defect. It will be appreciated that inany of the binding assays or the in vitro or in vivo functional assaysdescribed herein, a full-length MSC can be used, or, alternatively, afragment of an MSC can be used, for example a region containing only theankyrin repeats, containing only the transmembrane domains, containingonly the extracellular domain, or containing only a fragment of anythese regions, will be used. Further, such fragments can be used alone,or fused to a heterologous protein any other molecule.

DEFINITIONS

The term “mechanosensory transduction protein” refers to a polypeptidethat, when expressed in a cell or an oocyte, confers onto the cell anability to detect changes in pressure, motion, or any other mechanicalstimulus as described herein. Such proteins can be expressed naturallyor recombinantly, and can confer such activity on the cell in vitro, invivo, or ex vivo. Typically, such proteins will be at least about 70%identical to an amino acid sequence of SEQ ID NO:2, 4, or 6, and willinclude intracellular domains, including ankyrin repeats, andtransmembrane domains. However, such proteins can also refer to one ormore domains of these sequences in isolation, e.g., the ankyrin repeats,the extracellular domain, the transmembrane domains, or any subfragmentsthereof, alone. Such proteins can be involved in any mechanosensoryprocess, such as tactile sensation, proprioception, hearing,baroreception, and others.

The term “MSC protein” refers to polymorphic variants, alleles, mutants,and interspecies homologs that: (1) have about 70% amino acid sequenceidentity, preferably about 85-90% amino acid sequence identity to SEQ IDNOS:2, 4, or 6 over a window of about 25 amino acids, preferably 50-100amino acids; (2) bind to antibodies raised against an immunogencomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:2, 4, 6-9, and conservatively modified variants thereof; (3)specifically hybridize (with a size of at least about 500, preferably atleast about 900 nucleotides) under stringent hybridization and/or washconditions to a sequence selected from the group consisting of SEQ IDNO:1, 3, and 5, and conservatively modified variants thereof; or (4) areamplified by primers that specifically hybridize under stringenthybridization conditions to the same sequence as a degenerate primersets encoding SEQ ID NOS:7-9.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains an MSC protein or nucleic acid encoding an MSCprotein. Such samples include, but are not limited to, tissue isolatedfrom humans, mice, rats, and other animals. Biological samples may alsoinclude sections of tissues such as frozen sections taken forhistological purposes. A biological sample is typically obtained from aeukaryotic organism, such as insects, protozoa, birds, fish, reptiles,and preferably a mammal such as rat, mouse, cow, dog, guinea pig, orrabbit, and most preferably a primate such as chimpanzees or humans.Preferred tissues include tissues involved in mechanosensation, such asthe inner ear or any mechanosensory epithelial or neural tissue.

The phrase “functional effects” in the context of assays for testingcompounds that modulate MSC protein-mediated mechanosensory transductionincludes the determination of any parameter that is indirectly ordirectly under the influence of the channel. It includes changes in ionflux, membrane potential, current flow, transcription, MSC proteinphosphorylation or dephosphorylation, signal transduction, in vitro, invivo, and ex vivo and also includes other physiologic effects suchincreases or decreases of neurotransmitter or hormone release.

By “determining the functional effect” is meant assays for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of MSC proteins. Such functional effects can bemeasured by any means known to those skilled in the art, e.g., patchclamping, voltage-sensitive dyes, whole-cell currents, radioisotopeefflux, inducible markers, oocyte MSC expression; tissue culture cellMSC expression; transcriptional activation of MSC protein;ligand-binding assays; membrane potential and conductance changes;ion-flux assays; changes in intracellular calcium levels;neurotransmitter release, and the like.

A “physical effect” in the context of assays for testing the ability ofa compound to affect the activity of or bind to an MSC polypeptiderefers to any detectable alteration in the physical property or behaviorof an MSC polypeptide due to an interaction with a heterologouscompound, or any detection of a physical interaction using, e.g.,electrophoretic, chromatographic, or immunologically-based assay, orusing a two-hybrid screen as described infra. For example, a physicaleffect can include any alteration in any biophysical property of an MSCchannel comprising an MSC polypeptide, e.g., the cation specificity ormechanical sensitivity of the channel, or any structural or biochemicalproperties of an MSC polypeptide, e.g., its secondary, tertiary, orquaternary structure, hydrodynamic properties, spectral properties,chemical properties, or any other such property as described, e.g., inCreighton, Proteins (1984).

“Inhibitors,” “activators,” and “modulators” of MSC refer to anyinhibitory or activating molecules identified using in vitro and in vivoassays for mechanosensory transduction, e.g., agonists, antagonists, andtheir homologs and mimetics. Inhibitors are compounds that decrease,block, prevent, delay activation, inactivate, desensitize, or downregulate mechanosensory transduction, e.g., antagonists. Activators arecompounds that increase, open, activate, facilitate, enhance activation,sensitize or up-regulate mechanosensory transduction, e.g., agonists.Modulators include genetically-modified versions of MSC, e.g., withaltered activity, as well as naturally-occurring and synthetic ligands,antagonists, agonists, small chemical molecules and the like. Suchassays for inhibitors and activators include, e.g., expressing MSCprotein in cells or cell membranes, applying putative modulatorcompounds, and then determining the functional effects on mechanosensorytransduction, as described above. Samples or assays comprising MSC thatare treated with a potential activator, inhibitor, or modulator arecompared to control samples without the inhibitor, activator, ormodulator to examine the extent of inhibition. Control samples(untreated with inhibitors) are assigned a relative MSC activity valueof 100%. Inhibition of MSC is achieved when the C activity valuerelative to the control is about 80%, preferably 50%, more preferably25-1%. Activation of MSCs is achieved when the MSC activity valuerelative to the control is 110%, more preferably 150%, more preferably200-500%, more preferably 1000-3000% higher.

“Biologically active” MSC refers to an MSC protein, or a nucleic acidencoding the MSC protein, having mechanosensory transduction activity asdescribed above, involved in mechanosensory transduction inmechanosensory cells.

The terms “isolated” “purified” or “biologically pure” refer to materialthat is substantially or essentially free from components which normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high-performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. In particular, an isolated MSCnucleic acid is separated, e.g., from open reading frames or fragmentsof open reading frames, e.g., that naturally flank the MSC gene andencode proteins other than MSC protein. An isolated MSC nucleic acid istypically contiguous, i.e., heterologous sequences are typically notembedded in the MSC nucleic acid sequence, although heterologoussequences are often found ajoining an isolated MSC nucleic acidsequence. The term “purified” denotes that a nucleic acid or proteingives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at least 85%pure, more preferably at least 95% pure, and most preferably at least99% pure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group., e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes (A, T,G, C, U, etc.).

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which theany position of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues to yield a codon encoding thesame amino acid residue (Batzer et al., Nucleic Acid Res. 19:5081(1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossoliniet al., Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy ofthe genetic code, a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG and GCUall encode the amino acid alanine. Thus, at every position where analanine is specified by a codon in an amino acid herein, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants and allelesof the invention.

The following groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Serine (S), Threonine (T);

3) Aspartic acid (D), Glutamic acid (E);

4) Asparagine (N), Glutamine (Q);

5) Cysteine (C), Methionine (M);

6) Arginine (R), Lysine (K), Histidine (H);

7) Isoleucine (I), Leucine (L), Valine (V); and

8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984) for a discussion of amino acidproperties).

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins which can be madedetectable, e.g., by incorporating a radiolabel into the peptide or usedto detect antibodies specifically reactive with the peptide.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

As used herein, a “nucleic acid probe or oligonucleotide” is defined asa nucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Such sequencesare then said to be “substantially identical.” This definition alsorefers to the complement of a test sequence. Preferably, the percentidentity exists over a region of the sequence that is at least about 25amino acids in length, more preferably over a region that is 50 or 100amino acids in length.

For sequence comparison, one sequence acts as a reference sequence, towhich test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are entered into a computer,subsequence coordinates are designated, if necessary, and sequencealgorithm program parameters are designated. Default program parameterscan be used, or alternative parameters can be designated. The sequencecomparison algorithm then calculates the percent sequence identities forthe test sequences relative to the reference sequence, based on theprogram parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions,” or “stringent washconditions,” refers to conditions under which a probe will hybridize toits target subsequence, typically in a complex mixture of nucleic acid,but to no other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization. Washes can beperformed for varying amounts of time, e.g., 5 minutes, 15 minutes, 30minutes, 1 hour or more. Exemplary stringent hybridization or washconditions can be as following: 50% formamide, 5×SSC, and 1% SDS,incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with washin 0.2×SSC, and 0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions,” or“moderately stringent wash conditions,” include a hybridization in abuffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSCat 45° C. Washes can be performed for varying amounts of time, e.g., 5minutes, 15 minutes, 30 minutes, 1 hour or more. A positivehybridization is at least twice background. Those of ordinary skill willreadily recognize that alternative hybridization and wash conditions canbe utilized to provide conditions of similar stringency.

A further indication that two polynucleotides are substantiallyidentical is if the reference sequence, amplified by a pair ofoligonucleotide primers, can then be used as a probe under stringenthybridization and/or wash conditions to isolate the test sequence from acDNA or genomic library, or to identify the test sequence in, e.g., anorthern or Southern blot. Alternatively, another indication that thesequences are substantially identical is if the same set of PCR primerscan be used to amplify both sequences.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)−C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

An “anti-MSC” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the MSC gene, cDNA, or asubsequence thereof.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to MSC protein from specific species such as rat, mouse, or humancan be selected to obtain only those polyclonal antibodies that arespecifically immunoreactive with MSC and not with other proteins, exceptfor polymorphic variants and alleles of MSC. This selection may beachieved by subtracting out antibodies that cross-react with MSCproteins from other species. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, e.g., cultured cells, explants, and cells in vivo.

Isolation of MSC Nucleic Acids

General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter etal., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

Cloning MSC Nucleic Acids

In general, the nucleic acid sequences encoding MSC and related nucleicacid sequence homologs are cloned from cDNA and genomic DNA libraries byhybridization with a probe, or isolated using amplification techniqueswith oligonucleotide primers. For example, MSC sequences are typicallyisolated from mammalian nucleic acid (genomic or cDNA) libraries byhybridizing with a nucleic acid probe, the sequence of which can bederived from SEQ ID NOS:1, 3, or 5. MSC RNA and cDNA can be isolatedfrom any of a number of tissues, such as hair cells of the inner ear,sensory neurons, or any other mechanosensory cell.

Amplification techniques using primers can also be used to amplify andisolate an MSC polynucleotide from DNA or RNA. The degenerate primersencoding the following amino acid sequences can also be used to amplifya sequence of MSC: SEQ ID NOS:7-9 (see, e.g., Dieffenfach & Dveksler,PCR Primer: A Laboratory Manual (1995)). These primers can be used,e.g., to amplify either the full length sequence or a probe of one toseveral hundred nucleotides, which is then used to screen a mammalianlibrary for full-length MSC sequences.

Nucleic acids encoding MSC proteins can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using polypeptides comprising the sequence of,e.g., SEQ ID NOS:2, 4, 6, 7, 8 or 9.

cDNA and Genomic Libraries

MSC polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to MSC proteins can be isolated using MSCnucleic acid probes, and oligonucleotides under stringent hybridizationconditions, by screening libraries. Alternatively, expression librariescan be used to clone MSC and MSC polymorphic variants, alleles, andinterspecies homologs, by detecting expressed homologs immunologicallywith antisera or purified antibodies made against MSC, which alsorecognize and selectively bind to the MSC homolog.

To make a cDNA library, one should choose a source that is rich in MSCmRNA, e.g., inner ear tissue or other sources of mechanosensory cells,e.g., sensory epithelial cells or neurons. The mRNA is then made intocDNA using reverse transcriptase, ligated into a recombinant vector, andtransfected into a recombinant host for propagation, screening andcloning. Methods for making and screening cDNA libraries are well known(see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al.,supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

Amplification Methods

An alternative method of isolating MSC nucleic acid and its homologscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of MSCdirectly from mRNA, from cDNA, from genomic libraries or cDNA libraries.Degenerate oligonucleotides can be designed to amplify MSC homologsusing the sequences provided herein. Restriction endonuclease sites canbe incorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence ofMSC-encoding mRNA in physiological samples, for nucleic acid sequencing,or for other purposes. Genes amplified by the PCR reaction can bepurified from agarose gels and cloned into an appropriate vector.

Gene expression of MSC protein can be analyzed by techniques known inthe art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A⁺ RNA, Northern blotting, dot blotting,in situ hybridization, RNase protection, probing DNA microchip arrays,and the like. In one embodiment, high density oligonucleotide analysistechnology (e.g., GeneChip™) is used to identify homologs andpolymorphic variants of MSC. In the case where the homologs beingidentified are linked to a known disease, they can be used withGeneChip™ as a diagnostic tool in detecting the disease in a biologicalsample, see, e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14:869-876 (1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al.,Anal. Biochem. 224:110-106 (1995); Lockhart et al., Nat. Biotechnol.14:1675-1680 (1996); Gingeras et al., Genome Res. 8:435-448 (1998);Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998).

Synthetic oligonucleotides can be used to construct recombinant MSCgenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the MSC nucleic acid. The specificsubsequence is then ligated into an expression vector.

The nucleic acid encoding the MSC protein is typically cloned intointermediate vectors before transformation into prokaryotic oreukaryotic cells for replication and/or expression. These intermediatevectors are typically prokaryote vectors, e.g., plasmids, or shuttlevectors.

Expressing Nucleic Acids in Prokaryotes and Eukaryotes

Expression Vectors

To obtain high level expression of a cloned gene or nucleic acid, suchas those cDNAs encoding an MSC protein, one typically subclones MSC intoan expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the MSC protein areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kitsfor such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

Promoters

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the MSC-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding MSCprotein and signals required for efficient polyadenylation of thetranscript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding MSC protein may typically be linked to acleavable signal peptide sequence to promote secretion of the encodedprotein by the transformed cell. Such signal peptides would include,among others, the signal peptides from tissue plasminogen activator,insulin, and neuron growth factor, and juvenile hormone esterase ofHeliothis virescens. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

Other Elements

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a MSC encoding sequence underthe direction of the polyhedrin promoter or other strong baculoviruspromoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Transfection Methods

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of MSC protein,which are then purified using standard techniques (see, e.g., Colley etal., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingMSC.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofMSC, which is recovered from the culture using standard techniquesidentified below.

Purification of MSC Proteins

Either naturally occurring or recombinant MSC protein can be purifiedfor use in functional assays. Preferably, recombinant MSC is purified.Naturally occurring MSC is purified, e.g., from mammalian tissue such asinner ear tissue or other tissues including mechanosensory cells.Recombinant MSC is purified from any suitable expression system.

MSC protein may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant MSC is beingpurified. For example, proteins having established molecular adhesionproperties can be reversibly fused to MSC. With the appropriate ligand,MSC can be selectively adsorbed to a purification column and then freedfrom the column in a relatively pure form. The fused protein is thenremoved by enzymatic activity. Finally MSC could be purified usingimmunoaffinity columns.

Purification from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of MSCinclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. MSC is separated fromother bacterial proteins by standard separation techniques, e.g., withNi-NTA agarose resin.

Alternatively, it is possible to purify MSC protein from bacteriaperiplasm. After lysis of the bacteria, when MSC is exported into theperiplasm of the bacteria, the periplasmic fraction of the bacteria canbe isolated by cold osmotic shock in addition to other methods known toskill in the art. To isolate recombinant proteins from the periplasm,the bacterial cells are centrifuged to form a pellet. The pellet isresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria are centrifuged and the pellet is resuspended in ice-cold 5 mMMgSO₄ and kept in an ice bath for approximately 10 minutes. The cellsuspension is centrifuged and the supernatant decanted and saved. Therecombinant proteins present in the supernatant can be separated fromthe host proteins by standard separation techniques well known to thoseof skill in the art.

Standard Protein Purification Techniques

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of MSC protein can be used to isolated it fromproteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

MSC proteins can also be separated from other proteins on the basis ofits size, net surface charge, hydrophobicity, and affinity for ligands.In addition, antibodies raised against proteins can be conjugated tocolumn matrices and the proteins immunopurified. All of these methodsare well known in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

Affinity-Based Techniques

Any of a number of affinity based techniques can be used to isolate MSCproteins from cells, cell extracts, or other sources. For example,affinity columns can be made using anti-MSC antibodies or otherMSC-binding proteins, or physically-interacting proteins can beidentified by co-immunoprecipitation or other methods. Such methods arewell known to those of skill in the art and are taught, e.g., in Ausubelet al., Sambrook et al., Harlow and Lane, all supra.

Immunological Detection

In addition to the detection of MS genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect MSC proteins, e.g., to identify mechanosensory cells and variantsof MSC proteins. Immunoassays can be used to qualitatively orquantitatively analyze MSC proteins. A general overview of theapplicable technology can be found in Harlow & Lane, Antibodies: ALaboratory Manual (1988).

Antibodies to MSC Proteins

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with MSC proteins are known to those of skill in the art(see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow &Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2ded. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of MSC peptides or a full-length protein may be used to produceantibodies specifically reactive with MSC protein. For example,recombinant MSC protein, or an antigenic fragment thereof, is isolatedas described herein. Recombinant protein can be expressed in eukaryoticor prokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used as an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to MSC proteins. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see, Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-MSC proteinsor even other related proteins from other organisms, using a competitivebinding immunoassay. Specific polyclonal antisera and monoclonalantibodies will usually bind with a K_(d) of at least about 0.1 mM, moreusually at least about 1 μM, preferably at least about 0.1 μM or better,and most preferably, 0.01 μM or better.

Once MSC specific antibodies are available, MSC proteins can be detectedby a variety of immunoassay methods. For a review of immunological andimmunoassay procedures, see Basic and Clinical Immunology (Stites & Terreds., 7th ed. 1991). Moreover, the immunoassays of the present inventioncan be performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow &Lane, supra.

Immunological Binding Assays

MSC proteins can be detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241, 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case the MSC protein or antigenicsubsequence thereof). The antibody (e.g., anti-MSC) may be produced byany of a number of means well known to those of skill in the art and asdescribed above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled MSC polypeptide or alabeled anti-MSC antibody. Alternatively, the labeling agent may be athird moiety, such a secondary antibody, that specifically binds to theantibody/MSC complex (a secondary antibody is typically specific toantibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 11° C. to 40° C.

Non-Competitive Formats

Immunoassays for detecting MSC proteins in samples may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-MSC antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture MSC proteins present in the testsample. The MSC protein is thus immobilized and then bound by a labelingagent, such as a second MSC antibody bearing a label. Alternatively, thesecond antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety.

Competitive Formats

In competitive assays, the amount of MSC proteins present in the sampleis measured indirectly by measuring the amount of a known, added(exogenous) MSC proteins displaced (competed away) from an anti-MSCantibody by the unknown MSC protein present in a sample. In onecompetitive assay, a known amount of MSC protein is added to a sampleand the sample is then contacted with an antibody that specificallybinds to MSC proteins. The amount of exogenous MSC protein bound to theantibody is inversely proportional to the concentration of MSC proteinpresent in the sample. In a particularly preferred embodiment, theantibody is immobilized on a solid substrate. The amount of MSC proteinbound to the antibody may be determined either by measuring the amountof MSC protein present in a MSC protein/antibody complex, oralternatively by measuring the amount of remaining uncomplexed protein.The amount of MSC protein may be detected by providing a labeled MSCprotein molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known MSC protein, is immobilized on a solid substrate. Aknown amount of anti-MSC antibody is added to the sample, and the sampleis then contacted with the immobilized MSC protein. The amount ofanti-MSC antibody bound to the known immobilized MSC protein isinversely proportional to the amount of MSC protein present in thesample. Again, the amount of immobilized antibody may be detected bydetecting either the immobilized fraction of antibody or the fraction ofthe antibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

Cross-Reactivity Determination

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NOS:1, 3, or 5 can be immobilized to a solidsupport. Proteins (e.g., MSC proteins and homologs) are added to theassay that compete for binding of the antisera to the immobilizedantigen. The ability of the added proteins to compete for binding of theantisera to the immobilized protein is compared to the ability of MSCprotein encoded by SEQ ID NO:1, 3, or 5 to compete with itself. Thepercent crossreactivity for the above proteins is calculated, usingstandard calculations. Those antisera with less than 10% crossreactivitywith each of the added proteins listed above are selected and pooled.The cross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs. In one embodiment, antibodies thatcrossreact with MSC proteins from a different species are selectivelyremoved, thereby enhancing the species-specificity of the antisera. Forexample, to obtain antibodies that specifically react with DrosophilaMSC, the ability of SEQ ID NO:4 and SEQ ID NO:6 to compete for bindingto antisera directed against SEQ ID NO:4 are compared, and antibodiesthat cross-react with SEQ ID NO:6 selectively removed.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of MSC protein,to the immunogen protein (i.e., MSC protein of SEQ ID NOS:2, 4, 6-9). Inorder to make this comparison, the two proteins are each assayed at awide range of concentrations and the amount of each protein required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined. If the amount of the second protein required to inhibit 50%of binding is less than 10 times the amount of the protein encoded bySEQ ID NOS:1, 3, or 5 that is required to inhibit 50% of binding, thenthe second protein is said to specifically bind to the polyclonalantibodies generated to an MSC protein immunogen.

Other Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of MSC protein in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind MSC protein. The anti-MSC antibodies specificallybind to the MSC protein on the solid support. These antibodies may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the anti-MSC antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,Amer. Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly where theassay involves an antigen or antibody immobilized on a solid substrate,it is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used, with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecule (e.g., streptavidin) molecule,which is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize MSC protein, orsecondary antibodies that recognize anti-MSC protein.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Assays for Modulators of Mechanosensory Transduction

In numerous embodiments of this invention, assays will be performed todetect compounds that affect mechanosensory transduction in a cell. Suchassays can involve the identification of compounds that interact withMSC proteins, either physically or genetically, and can thus rely on anyof a number of standard methods to detect physical or geneticinteractions between compounds. Such assays can also involve thedetection of mechanosensory transduction in a cell or cell membrane,either in vitro or in vivo, and can thus involve the detection oftransduction activity in the cell through any standard assay, e.g., bymeasuring ion flux, changes in membrane potential, and the like. Suchcell-based assays can be performed in any type of cell, e.g., a sensorycell that naturally expresses MSC, a cultured cell that produces MSC dueto recombinant expression, or, preferably, an oocyte that is induced toproduce MSC through any of a number of means, as described infra.

In any of the binding or functional assays described herein, in vivo orin vitro, any MSC protein, or any derivative, variation, homolog, orfragment of an MSC protein, can be used. Preferably, the MSC protein isat least about 70% identical to SEQ ID NO:2, 4, or 6, and/or comprisesSEQ ID NO:7, 8, or 9. In numerous embodiments, a fragment of an MSCprotein is used. For example, a fragment that contains only theextracellular region, the ankyrin repeat region, or the transmembranedomains, i.e. the channel region (see, e.g., SEQ ID NOs:10-17), can beused. Such fragments can be used alone, in combination with other MSCfragments, or in combination with sequences from a heterologous protein,e.g., the fragments can be fused to a heterologous polypeptide, therebyforming a chimeric polypeptide. Any individual domain or sequence,however small, can readily be used in the present invention, e.g., asingle ankyrin repeat, transmembrane domain, etc., alone or incombination with other domains or with sequences from heterologousproteins. Such fragments and isolated domains of MSC proteins comprisean essential aspect of the present invention, and are of substantialimportance in the assays described herein.

Assays for MSC-Interacting Compounds

In certain embodiments, assays will be performed to identify moleculesthat physically or genetically interact with MSC proteins. Suchmolecules can be any type of molecule, including polypeptides,polynucleotides, amino acids, nucleotides, carbohydrates, lipids, or anyother organic or inorganic molecule. Such molecules may representmolecules that normally interact with MSC to effect mechanosensation insensory cells, or may be synthetic or other molecules that are capableof interacting with MSC and which can potentially be used to modulateMSC activity in cells, or used as lead compounds to identify classes ofmolecules that can interact with and/or modulate MSC. Such assays mayrepresent physical binding assays, such as affinity chromatography,immunoprecipitation, two-hybrid screens, or other binding assays, or mayrepresent genetic assays as described infra.

Such interacting molecules may interact with any part of an MSC protein,e.g., the extracellular domain, the transmembrane domain region, or theintracellular domain, including the ankyrin repeats. MSC proteins act insensory cells to depolarize the cell in response to a mechanical inputoutside of the cell. As such, interacting molecules may include thosethat interact with the extracellular domain of the protein, and whichmay enhance, inhibit, or otherwise modulate the detection of amechanical input, and which may be part of, or interact with, anextracellular structure involved in mechanical detection, such as thestereocilium of a hair cell. An interacting molecule may also interactwith the transmembrane domain region of the protein, and may be involvedin, or capable of modulating, the formation of a channel, the opening orclosing of a channel, etc. In addition, an interacting molecule mayinteract with an intracellular part of a channel, e.g., an ankyrinrepeat, and be involved in, e.g., the function, regulation, adaptation,or any other aspect of channel activity.

The MSC protein used in such assays can be a full-length MSC protein orany subdomain of an MSC protein. In preferred embodiments, a fragment ofan MSC protein comprising an extracellular domain of an MSC will beused. Molecules that bind to the extracellular domain of an MSC areparticularly useful for the identification of modulators of MSCactivity, as they are typically soluble and readily included in highthroughput screening assay formats, as described infra.

Assays for Physical Interactions

Compounds that interact with MSC proteins can be isolated based on anability to specifically bind to an MSC protein or fragment thereof. Innumerous embodiments, the MSC protein or protein fragment will beattached to a solid support. In one embodiment, affinity columns aremade using the MSC polypeptide, and physically-interacting molecules areidentified. It will be apparent to one of skill that chromatographictechniques can be performed at any scale and using equipment from manydifferent manufacturers (e.g., Pharmacia Biotech). In addition,molecules that interact with MSC proteins in vivo can be identified byco-immunoprecipitation or other methods, i.e. immunoprecipitating MSCproteins using anti-MSC antibodies from a cell or cell extract, andidentifying compounds, e.g., proteins, that are precipitated along withthe MSC protein. Such methods are well known to those of skill in theart and are taught, e.g., in Ausubel et al., Sambrook et al., Harlow &Lane, all supra.

Two-hybrid screens can also be used to identify polypeptides thatinteract in vivo with an MSC or a fragment thereof (Fields et al.,Nature 340:245-246 (1989)). Such screens comprise two discrete, modulardomains of a transcription factor protein, e.g., a DNA binding domainand a transcriptional activation domain, which are produced in a cell astwo separate polypeptides, each of which also comprises one of twopotentially binding polypeptides. If the two potentially bindingpolypeptides in fact interact in vivo, then the DNA binding and thetranscriptional activating domain of the transcription factor areunited, thereby producing expression of a target gene in the cell. Thetarget gene typically encodes an easily detectable gene product, e.g.,β-galactosidase, which can be detected using standard methods. In thepresent invention, an MSC polypeptide is fused to one of the two domainsof the transcription factor, and the potential MSC-binding polypeptides(e.g., encoded by a cDNA library) are fused to the other domain. Suchmethods are well known to those of skill in the art, and are taught,e.g., in Ausubel et al., supra.

Assays for Genetic Interactions

It is expected that MSCs are assembled into multi-protein complexes inwhich the interactions are mediated by the large number of ankyrinrepeats found in the N terminus of the protein. Genetic screens can thusbe performed to identify such additional proteins that are involved inthe transduction pathway. For example, genetic strains are produced thatpossess only a partially functional nompC (MSC) gene, which confers anincomplete mechanical sensitivity to the fly. Ideally, a vial of theseflies would produce only 10-20 viable homozygotes. In this sensitizedgenetic background, flies will be screened for mutations in other genesthat either suppress or enhance the survival of the mutant flies. Flieswill be mutagenized using any standard chemical, radiation-based, orgenetic method and then crossed into the above-described sensitizedgenetic background, followed by counting the number of homozygousprogeny. Mutations that produce more than 10-20 flies per vial areconsidered suppressors of nompC, and those that produce fewer flies areconsidered enhancers. Similar screens can be performed using MSC genesin genetically tractable mammals, e.g., mice.

Assays for MSC Activity

The activity of MSC polypeptides, and any homolog, variant, derivative,or fragment thereof can be assessed using a variety of in vitro and invivo assays for mechanoreceptor potential, e.g., measuring current,measuring membrane potential, measuring ion flux, e.g., potassium orcalcium, measuring transcription levels, measuring neurotransmitterlevels, using e.g., voltage-sensitive dyes, radioactive tracers,patch-clamp electrophysiology, transcription assays, and the like.Furthermore, such assays can be used to test for modulators, e.g.,inhibitors or activators, of MSC. Such modulators can be a protein,amino acid, nucleic acid, nucleotide, lipid, carbohydrate, or any typeof organic or inorganic molecule, including genetically altered versionsof MSC proteins. Such assays can be performed using any of a largenumber of cells, including oocytes, cultured cells, sensory epithelialor neural cells, and others, and can be present in vitro or in vivo.Such cells can contain naturally expressed MSC, can be induced toexpress MSC using recombinant or other methods, or can comprise MSC bydirect addition of the protein to the cell or cell membrane. In numerousembodiments, the cell or cell membrane comprising the MSC polypeptidewill be anchored to a solid support.

Preferably, the MSC proteins used in the assay is selected from apolypeptide having a sequence of SEQ ID NOS:2, 4, or 6, or aconservatively modified variant thereof. Alternatively, the MSC proteinused in the assay will be derived from a eukaryote and include an aminoacid subsequence having amino acid sequence identity SEQ ID NOS:2, 4, or6. Generally, the amino acid sequence identity will be at least 70%,preferably at least 85%, most preferably at least 90-95%. In preferredembodiments, a polypeptide comprising an extracellular domain is used,e.g., an extracellular domain of SEQ ID NO:2, 4, or 6. In suchembodiments, the extracellular domain is often fused to a heterologouspolypeptide, forming a chimeric polypeptide. Typically, such chimericpolypeptides will comprise an extracellular domain as well as multipletransmembrane domains, and will have mechanosensory transductionactivity.

Detecting Mechanosensory Transduction

In numerous embodiments of the present invention, assays will beperformed to detect alterations in an MSC protein, e.g., one expressedin a cell or cell membrane, or in mechanosensory transduction, ormechanoreceptor potential, in a cell or cell membrane, e.g., as a resultof a mutation in an MSC or due to the presence of an MSC-modulatingcompound. Mechanosensory transduction or mechanoreceptor potential canbe detected in any of a number of ways, including by detecting changesin ion flux, changes in polarization of a cell or cell membrane, changesin current, and other methods, including by measuring downstreamcellular effects, e.g., neuronal signaling.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing MSC. One means to determine changes in cellular polarizationis by measuring changes in current (thereby measuring changes inpolarization) with voltage-clamp and patch-clamp techniques, e.g., the“cell-attached” mode, the “inside-out” mode, and the “whole cell” mode(see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)).Whole cell currents are conveniently determined using the standardmethodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85 (1981).Other known assays include: radioactive ion flux assays and fluorescenceassays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind etal., J. Membrane Biol. 88:67-75 (1988); Gonzales & Tsien, Chem. Biol.4:269-277 (1997); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991);Holevinsky et al., J. Membrane Biology 137:59-70 (1994)). Generally,candidate compounds are tested in the range from 1 pM to 100 mM.

The effects of the test compounds, or sequence variation, upon thefunction of the MSC polypeptides can be measured by examining any of theparameters described above. In addition, any suitable physiologicalchange that affects MSC activity, or reflects MSC activity, can be usedto assess the influence of a test compound or sequence alteration on theMSC polypeptides of this invention. When the functional consequences aredetermined using intact cells or animals, one can also measure a varietyof effects such as transmitter release, hormone release, transcriptionalchanges to both known and uncharacterized genetic markers (e.g.,northern blots), changes in cell metabolism such as cell growth or pHchanges, and other effects.

Preferred assays for mechanosensory transduction channels include cells,e.g., oocytes, that are loaded with ion or voltage sensitive dyes toreport receptor activity. Assays for determining activity of suchreceptors can also use known agonists and antagonists for other cationchannels as negative or positive controls to assess activity of testedcompounds. In assays for identifying modulatory compounds (e.g.,agonists, antagonists), changes in the level of ions in the cytoplasm ormembrane voltage will be monitored using an ion-sensitive or membranevoltage fluorescent indicator, respectively. Among the ion-sensitiveindicators and voltage probes that may be employed are those disclosedin the Molecular Probes 1997 Catalog. In addition, changes incytoplasmic calcium, potassium, or other ion levels can be used toassess MSC function.

In vivo Assays

In certain embodiments, the mechanosensory activity of a cell will beexamined in vivo. Such embodiments are useful for, e.g., examining theactivity of an MSC or an MSC mutant, derivative, homolog, fragment, etc.Also, such assays are useful for detecting the activity of candidate MSCmodulator in vivo. Potential MSCs can be produced in transgenic fliescarrying the candidate cDNA driven by a suitable, e.g. a nompC,promoter/enhancer construct. These candidate channels can be expressedin mechanosensory neurons of flies and their mechanoelectrical activitymeasured with bristle recordings. Methods of producing transgenic fliesand methods of detecting mechanosensory transduction activity in flymechanosensory neurons are well known to those of skill in the art andare described, e.g., in Drosophila, a Practical Approach (Roberts, ed.1986)), and in Kernan et al. (1994), respectively.

Alternatively, it is possible to screen for molecules that can mimicNOMPC activity by performing the screen in a nompC mutant background.Those molecules that rescue the mutant phenotype can be consideredpotential MSCs.

Assays Using Oocytes or Cultured Cells in vitro

Xenopus oocytes

In preferred embodiments, MSC proteins are expressed in oocytes of thefrog Xenopus laevis, and the mechanosensory transduction of the oocytemeasured. Such assays are useful, e.g., to measure the activity ofhomologs, variants, derivatives, and fragments of MSC proteins, as wellas to measure the effect of candidate modulators on the activity of MSCprotein channels in the oocytes. In such embodiments, mRNA encoding theMSC protein, or candidate MSC protein, is typically microinjected intothe oocyte where it is translated. The MSC protein, and in some casesthe candidate MSC, then forms a functional mechanosensory transductionchannel in the oocyte which can be studied using the methods describedherein. In such embodiments, MSC cDNAs are typically subcloned intospecialized transcription vectors in which the cDNA insert is flanked byXenopus hemoglobin 5′ and 3′ untranslated regions. Transcripts are madefrom both the sense and antisense strand of the plasmid and thenpolyadenylated using standard techniques. These transcripts are thenmicroinjected into Xenopus laeves oocytes. After allowing a sufficienttime for translation, the oocytes are subjected to voltage-clamprecording. Cell-attached patches of oocyte membrane are assayed for thepresence of conductances provoked by the application of mechanical forceto the membrane, e.g., using small, calibrated pressure and vacuum stepsapplied through the patch pipette. Because Xenopus oocyte membranescontain an endogenous mechanically gated conductance, which is typicallyobserved using these methods, the conductance due to the heterologousMSC channel represents any additional conductance, i.e., beyond thebackground level, seen during a mechanical stimulus. In such assays, itis important to compare the sense- to the antisense- and mock-injectedcontrols for the presence of mechanically gated conductances.

Cultured Cells

In certain embodiments, MSC proteins are expressed in cultured cells,e.g., mammalian cells, and the mechanosensory transduction activity ofthe cell determined. In such assays, cDNAs encoding known or candidateMSC proteins are typically subcloned into commercially available cellexpression vectors, e.g., mammalian cell expression vectors, and thentransfected into cultured cells. Expression vectors, transfection, andmaintenance of animal cells are well known to those of skill and aretaught, e.g., in Ausubel et al., supra, and Freshney, The Culture ofAnimal Cells (1993).

Cultured animal cells expressing MSC proteins, like the above-describedoocytes, are subjected to cell-attached patch voltage-clamp recordingduring the application of mechanical stimuli such as small, calibratedpressure and suction stimuli to the patch. Osmotic membrane stress canalso serve as a mechanical stimulus. Again, as eukaryotic cellsgenerally contain endogenous mechanically gated ion channels, it isimportant to compare the transduction levels in the transfected cells tothose in the mock-transfected controls. Any mechanically-gatedconductance detectable above the level of the endogenous conductance isdue to the candidate channel.

Alternatively, because MSC channels conduct calcium ions, transfectedcells are loaded with a fluorescent Ca²⁺ indicator dye and thenstimulated with hypo- and hyper-osmotic solutions while monitoring thecell's fluorescence. Hyper- and hypo-osmotic solutions create membranestresses that open mechanically gated ion channels. In such assays, theinflux of Ca²⁺ causes an increase in fluorescence of the Ca²⁺ indicatordye. As with the voltage-clamp recording, it is important to compare thetransfected and mock-transfected controls. Any increased fluorescence inthe transfected cells during the stimuli compared to that observed inmock transfected cells is due to the presence of the MSC channel.

Biophysical Properties of MSC Channels

The effect of a sequence alteration in an MSC channel, or of a candidatemodulator on a channel, can also be assessed by examining the effect ofthe sequence alteration or the compound on one or more structural orbiophysical properties that are typical of MSC channels. For example,MSC channels show very little voltage dependence, and are instead gatedby mechanical stimuli. Further, MSC channels have a non-specificcationic preference, i.e., they conduct many different cations,including some large organic cations like tetramethyl ammonium ion(although weakly). The solution bathing these channels in the Drosophilabristle and in vertebrate hearing organs has a high potassium ionconcentration (over 100 mM), which is very unusual for an extracellularfluid. Because of this, the principal current-carrying ion in vivo isK⁺, with a small portion of the current carried by Ca²⁺. In addition, asMSC channels are completely blocked in vivo by tetraethyl ammonium ions,it is expected that the channels are also refractory to tetraethylammonium ions in heterologous systems. Further, MSC proteins are ingeneral refractory to Gd³⁺ ions, albeit at millimolar concentrations; inour bristle recording system, however, fly mechanoreceptor neurons areunaffected by Gd³⁺ treatment.

It will be appreciated that any of these characteristics, which aretypical of mechanosensory transduction channels in vivo, can be assessedin cell-attached patches in either oocytes or cultured cells to assessthe effect of any potential modulator, mutation, or treatment upon anMSC protein.

Candidate Modulators and MSC-Binding Compounds

Using the present methods, any protein, amino acid, nucleic acid,nucleotide, carbohydrate, lipid, or any other organic or inorganicmolecule can be assessed for its ability to bind to or modulate theactivity of an MSC polypeptide. Such candidate modulators or bindingproteins can be deliberately designed, e.g., a putativedominant-negative form of an MSC polypeptide or a compound predicted tobind based on a computer-based structural analysis of the protein, orcan be identified using high efficiency assays to rapidly screen a largenumber of potential compounds, e.g., from a library of nucleic acids ora combinatorial peptide or chemical library.

Proteins

Any of a number of polypeptides can be used in the present assays todetermine their ability to bind to or modulate mechanosensorytransduction activity in an MSC-protein expressing cell. Suchpolypeptides can represent, e.g., a candidate protein or collection ofproteins encoded by a library of nucleic acids, can represent a putativedominant negative form or other variant of an MSC polypeptide, canrepresent a collection of peptide sequences, e.g., from a combinatorialpeptide library, or can be predicted using a computer-based structuralanalysis program.

Heterologous Proteins

Polypeptide modulators of MSC proteins can be identified using afluorescence-based screening strategy. In such approaches, cells arefirst induced to stably express an MSC protein, and then transfectedwith a cDNA clone of interest, e.g., representing adeliberately-selected candidate modulator or a collection of randomclones such as a cDNA library isolated from a sensory tissue. Thetransfected cells are then loaded with fluorescent Ca²⁺-indicator dyesand subjected to an osmotic stimulus or a mild mechanical treatment.Heterologous proteins that exert a modulatory effect on the MSC channelwill cause the cell to exhibit either an increase or a decrease in thefluorescence during the stimulus compared to a cell expressing the MSCprotein alone.

MSC Protein Fragments. e.g. Dominant Negative Forms

Because MSCs are thought be part of a multi-protein complex in vivo, itis expected that a dominant-negative form of MSC can be produced bydesigning an MSC that lacks mechanosensory transduction activity butwhich can nevertheless interact in vivo with other molecules involved inmechanosensory transduction. A “dominant-negative” MSC refers to any MSCwhose presence reduces mechanosensory activity in vivo, even in thepresence of fully functional MSC protein. For example, overexpression ofthe ankyrin repeats alone (which are thought to facilitateprotein-protein interactions), or in combination with a defectivechannel domain, will likely lead to the disruption of mechanicalsignaling. Alternatively, if these channels are comprised of severalhomomeric subunits (e.g., single MSC polypeptide units), expression ofthe channel moiety alone will reduce mechanosensory signaling in adominant fashion.

In addition, because MSCs are weakly similar at a structural level tomany voltage-activated channels, they could potentially contain anendogenous “ball and chain” inactivator of the channel (see, e.g., Antzet al., Nat Struct Biol 6(2):146-50 (1999)). Accordingly, one canpotentially identify such endogenous modulators by producing smallfragments of MSC, e.g., using a bacterial expression system, andassaying their ability to inhibit MSC protein activity in an assay asdiscussed supra.

Small Molecules

In numerous embodiments of this invention, test compounds will be smallchemical molecules or peptides. Essentially any chemical compound can beused as a potential modulator or ligand in the assays of the invention,although most often compounds that can be dissolved in aqueous ororganic (especially DMSO-based) solutions are used. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source to assays,which are typically run in parallel (e.g., in microtiter formats onmicrotiter plates in robotic assays). It will be appreciated that thereare many suppliers of chemical compounds, including Sigma (St. Louis,Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs Switzerland) and the like.

Combinatorial Libraries

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

High Throughput Screening

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell, cell membrane, ortissue comprising the MSC protein is attached to a solid phasesubstrate. In the high throughput assays of the invention, it ispossible to screen up to several thousand different modulators orligands in a single day. In particular, each well of a microtiter platecan be used to run a separate assay against a selected potentialmodulator, or, if concentration or incubation time effects are to beobserved, every 5-10 wells can test a single modulator. Thus, a singlestandard microtiter plate can assay about 100 (e.g., 96) modulators. If1536 well plates are used, then a single plate can easily assay fromabout 100- about 1500 different compounds. It is possible to assayseveral different plates per day; assay screens for up to about6,000-20,000 different compounds is possible using the integratedsystems of the invention. More recently, microfluidic approaches toreagent manipulation have been developed.

Computer-Based Assays

Yet another assay for compounds that modulate MSC activity involvescomputer assisted drug design, in which a computer system is used togenerate a three-dimensional structure of MSC proteins based on thestructural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apre-established algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind heterologous molecules. Theseregions are then used to identify molecules that bind to the protein.

The three-dimensional structural model of the protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a MSC polypeptide intothe computer system. For example, the amino acid sequence of thepolypeptide is selected from the group consisting of SEQ ID NOS:2, 4,and 6, and conservatively modified versions thereof. The amino acidsequence represents the primary sequence or subsequence of the protein,which encodes the structural information of the protein. At least 10residues of the amino acid sequence (or a nucleotide sequence encoding10 amino acids) are entered into the computer system from computerkeyboards, computer readable substrates that include, but are notlimited to, electronic storage media (e.g., magnetic diskettes, tapes,cartridges, and chips), optical media (e.g., CD-ROM), informationdistributed by internet sites, and by RAM. The three-dimensionalstructural model of the protein is then generated by the interaction ofthe amino acid sequence and the computer system, using software known tothose of skill in the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential binding regions areidentified by the computer system. Three-dimensional structures forpotential binding molecules are generated by entering amino acid ornucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential binding moleculeis then compared to that of the MSC protein to identify molecules thatbind to MSC. Binding affinity between the protein and binding moleculeis determined using energy terms to determine which molecules have anenhanced probability of binding to the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of MSC genes. Such mutationscan be associated with disease states or genetic traits. As describedabove, GeneChip™ and related technology can also be used to screen formutations, polymorphic variants, alleles and interspecies homologs. Oncethe variants are identified, diagnostic assays can be used to identifypatients having such mutated genes. Identification of the mutated MSCprotein encoding genes involves receiving input of a first nucleic acidor amino acid sequence encoding MSC proteins, e.g., a sequence selectedfrom the group consisting of SEQ ID NOS:1-9, and conservatively modifiedversions thereof. The sequence is entered into the computer system asdescribed above. The first nucleic acid or amino acid sequence is thencompared to a second nucleic acid or amino acid sequence that hassubstantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in MSC protein encoding genes, andmutations associated with disease states and genetic traits.

MSC Genotyping

The present invention also provides methods to genotype an animal,including a human, for an MSC gene or protein. Typically, suchgenotyping involves a determination of the particular sequence, allele,or isoform of an MSC gene or protein, using any standard technique asdescribed herein, including DNA sequencing, amplification-based,restriction enzyme-based, electrophoretic and hybridization based assaysto detect variations in genomic DNA or mRNA, or immunoassays andelectrophoretic assays to detect protein variations. The detection ofparticular alleles, sequence variations, isoforms, etc., is useful formany applications, including for forensic, paternity, epidemiological,or other investigations.

In addition, the detection of certain alleles or protein forms is usefulfor the detection of a mutation in an MSC gene in an animal, and is thususeful for the diagnosis of mechanosensory transduction channel defectsin the animal. Such mechanosensory defects may underlie any of a largevariety of conditions in animals, including conditions associated withimpaired hearing, touch sensitivity, proprioception, balance, and otherprocesses. In addition, mechanosensory defects may be associated with aloss of contact-inhibition in cells, and thus may be associated withcancer in the animal.

In particular, it has been discovered that mutations that introduce apremature stop codon into an MSC gene within the ankyrin repeat region,or mutations that remove or substitute a conserved cysteine residuebetween transmembrane segments 4 and 5 of the protein, result in adramatic decrease in MSC activity and are thus useful markers for suchanalyses.

Pharmaceutical Compositions and Administration

Mechanosensory transduction modulators can be administered directly tothe mammalian subject for modulation of mechanosensation in vivo.Administration is by any of the routes normally used for introducing amodulator compound into ultimate contact with the tissue to be treated,such as the inner ear or other mechanosensory tissue. The mechanosensorymodulators are administered in any suitable manner, preferably withpharmaceutically acceptable carriers. Suitable methods of administeringsuch modulators are available and well known to those of skill in theart.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985))

Kits

MSC proteins and their homologs are useful tools for identifyingmechanosensory cells, for forensics and paternity determinations, forexamining mechanosensory transduction, and for diagnosing mechanosensorydefects in animals. MSC specific reagents that specifically hybridize toMSC protein-encoding nucleic acid, such as MSC specific probes andprimers, and MSC specific reagents that specifically bind to the MSCprotein, e.g., MSC specific antibodies are used to examinemechanosensory cell expression and mechanosensory transductionregulation.

Nucleic acid assays for the presence of MSC encoding DNA and RNA in asample include numerous techniques are known to those skilled in theart, such as Southern analysis, northern analysis, dot blots, RNaseprotection, S1 analysis, amplification techniques such as PCR and LCR,and in situ hybridization. In in situ hybridization, for example, thetarget nucleic acid is liberated from its cellular surroundings in sucha way as to be available for hybridization within the cell whilepreserving the cellular morphology for subsequent interpretation andanalysis. The following articles provide an overview of the art of insitu hybridization: Singer et al., Biotechniques 4:230-250 (1986); Haaseet al., Methods in Virology, vol. VII, pp. 189-226 (1984); and NucleicAcid Hybridization: A Practical Approach (Hames et al., eds. 1987). Inaddition, MSC protein can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing recombinant MSCprotein) and a negative control.

The present invention also provides for kits for screening formodulators of MSC proteins. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: MSC protein, reaction tubes,and instructions for testing MSC activity. Preferably, the kit containsbiologically active MSC protein. A wide variety of kits and componentscan be prepared according to the present invention, depending upon theintended user of the kit and the particular needs of the user.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to one ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

EXAMPLES Example I Chromosome Mapping and Positional Cloning of MSCGenomic Region

To identify mutations with potential roles in mechanosensorytransduction, a genetic screen was carried out to identify mutations inDrosophila melanogaster that result in uncoordination phenotypes. Thisscreen yielded mutations in numerous genes. Further characterization ofthese mutations using electrophysiological methods determined thatseveral of the genes also reduced or eliminated bristle mechanoreceptorpotentials (Kernan et al., Neuron 12:1195-1206 (1994)). One of thesemutations, responsible for the nompC (for no-mechanoreceptor potential),present on the second chromosome, abolished nearly all of themechanoelectrical transduction in mutant cells. Flies with this mutationare uncoordinated to the point of lethality. Based on these phenotypes,the gene underlying the nompC mutant was identified as potentiallyencoding a protein playing a central role in mechanosensorytransduction, such as a mechanosensory transduction channel.

To determine the position of the nompC gene on the second chromosome,nompC mutations were genetically combined with various secondchromosomal deletions, and the resulting transheterozygous flies werescreened for lethality. In this way, the chromosomal position of thenompC mutation was mapped to a small interval on the left arm of thesecond chromosome, corresponding to map positions 25D6-7.

To physically isolate DNA in the 25D6-7 region, the proximal-most clonefrom a chromosomal walk in the nearby 25D1-4 region (George & Terracol,Genetics 146:1345-1363 (1997)) was used to probe a Drosophila cosmidlibrary (Tamkun et al., 1992). Overlapping clones were used to “walk” tothe area that contained the nompC (MSC) protein encoding gene, bymapping the cosmid clones to genetic breakpoints. At the same time, thecosmids were tested for the ability to rescue the nompC mutantphenotype. One cosmid was found to rescue the lethality, uncoordinatedbehavior, and physiological defect of the nompC mutation. This cosmidwas thus determined to likely contain the MSC protein-encoding gene.

Example II Sequencing of the Rescuing Cosmid and MSC Gene

To determine the sequence of the cosmid containing the MSC proteinencoding gene, the genomic DNA insert from the cosmid was isolated,sonicated, polished, size-selected, and the resulting 0.7-2 kb fragmentssubcloned into plasmid vectors. Plasmids were purified and analyzed forthe presence and size of inserts, and 123 clones with inserts of greaterthan 0.7 kb were sequenced. The sequences determined from these insertswere used to assemble large contiguous fragments, which were extended bydesigning ad hoc primers from the ends of the fragments and using theprimers to read additional sequence from the cosmid DNA. In this way,the entire 33.6 kb cosmid insert was sequenced.

The MSC protein-encoding gene was identified and characterized withinthis 33.6 kb cosmid sequence using exon analysis, BLAST searches, andsecondary-structure prediction programs. These analyses established thatthe MSC gene is a large gene comprised of 19 exons, encoding a proteincontaining at least 21 ankyrin repeats and a set of as many as 11transmembrane domains (6 of which show significant robustness), that isweakly related to the TRP family of epithelial cation channels (see, forexample, Montell, Curr Opin Neurobiol 8:389-97 (1988)).

Example III Sequencing of nompC Mutants

To assess the molecular defects of the nompC mutants, we used PCR toamplify the genomic DNA encompassing the nompC locus from flies with oneof four mutant nompC alleles. In this way, all four alleles of the nompCgene were amplified in approximately 2 kb fragments that covered thegene interval. These fragments were then sequenced. All four of thenompC alleles showed mutations in the coding region when compared to thesequence of the cosmid and to the parental, wild type DNA.

In three of these alleles, the nompC (MSC) polypeptide encoded by themutant gene was prematurely truncated in the ankyrin repeats by theintroduction of stop codons. The fourth allele had a missense mutationbetween transmembrane segments four and five, resulting in a C to Ysubstitution.

Example IV Identifying MSC-Related Genes in Other Organisms

To identify potential MSC-related genes in other organisms, we performedsequence comparisons between Drosophila MSC sequences and nucleotideand/or amino acid sequences present in various public databases. In thisway, a previously unknown C. elegans genomic sequence was identified asan MSC homolog. This genomic fragment was found in the“unfinished/orphan” domain of the C. elegans genome project database.Using a variety of sequence analysis programs, putative coding exons,intron sequences, candidate transmembrane domains, and homology regionswith Drosophila MSC were identified. FIG. 1 shows an alignment betweenthe Drosophila melanogaster and C. elegans MSC homologs.

Three signature sequences for MSC, based on alignment analysis betweenthe Drosophila and C. elegans sequences, were identified and are shownas SEQ ID NOs:7, 8, and 9.

1-17. (canceled)
 18. A method for identifying a compound that modulatesmechanosensory receptor activity in eukaryotic cells, the methodcomprising the steps of: (i) contacting the compound with amechanosensory receptor protein, the protein having at least one of thefollowing characteristics: (a) comprising greater than about 70% aminoacid sequence identity to a sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; (b) comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:7, SEQ IDNO:8, and SEQ ID NO:9; or (c) specifically binding to polyclonalantibodies generated against a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,and SEQ ID NO:6; and (ii) determining the functional effect of thecompound on the mechanosensory receptor protein.
 19. The method of claim18, wherein the mechanosensory receptor protein is expressed in aeukaryotic cell or cell membrane.
 20. The method of claim 19, whereinthe functional effect is determined by detecting a change in themechanoreceptor potential of the cell or cell membrane.
 21. The methodof claim 19, wherein the functional effect is determined by detecting achange in an intracellular ion concentration.
 22. The method of claim21, wherein the ion is selected from the group consisting of K⁺ andCa²⁺.
 23. The method of claim 18, wherein the protein comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, and SEQ ID NO:6.
 24. The method of claim 18, wherein the proteinis recombinant.
 25. The method of claim 18, wherein the functionaleffect is a physical interaction with the receptor protein. 26.(canceled)